Project Summary Activation of the zygotic genome is a landmark in development, and yet how this happens is not fully understood. Recently a new concept has emerged of ?limited machinery? where maternal regulation of the accumulation of specific transcription factors contributes to the timing of zygotic genome activation. In zebrafish, one of these transcription factors is Pou5. This project will test if Pou5 also activates the genome in another vertebrate species using Xenopus frogs. A little-known family of maternal factors, Zar1 (Zygote arrest 1) and Zar2, that are crucial for development, has been implicated in zygotic genome activation in mice. Xenopus Zar1 and Zar2 bind to maternal mRNAs, including Pou5, and regulate translation of mRNA reporters. This evidence supports the hypotheses that 1) Zar1 and/or Zar2 bind to Pou5 mRNA and repress translation to prevent accumulation of Pou5 protein in oocytes that in turn prevents premature transcription; and 2) Zar1 and/or Zar2 bind to Pou5 mRNA and increase translation leading to accumulation of Pou5 protein in embryos and activation of zygotic transcription. To test if the accumulation of Pou5 protein in oocytes and/or embryos is dependent on Zar1 and/or Zar2 function, newly developed interfering mutants of Zar1 and Zar2 will be used. As Zar1 and Zar2 bind to cis-elements in the 3' untranslated region (UTR) of maternal mRNAs, the 3' UTR of Pou5 will be fused to a luciferase reporter to test if it mediates the translational regulation. The Zar1 and Zar2 interfering mutants will also be used to test if transcription in oocytes or embryos is dependent on Zar1 and/or Zar2 function. Antisense morpholinos will functionally knockdown Pou5 to test if zygotic transcription is dependent on Pou5 function. In many of these experiments, manipulation of protein expression in embryos will be performed using the host transfer technique. The advantage of this approach is that the frog egg already contains interfering mutants and/or morpholinos before fertilization, so their effects on the very earliest cleavage stages of embryogenesis can be assayed. Because Pou5 is not expressed in the entire Xenopus embryo, the last hypothesis to be tested is that Zar1 and/or Zar2 bind to other mRNAs encoding transcription factors, that regulate zygotic genome activation in other regions of the embryo. This will be tested by isolating RNAs that co-purify with Zar1 and Zar2 and identifying them by RNAseq. This will generate a list of mRNAs that will be studied in the future. These studies will reveal whether the Pou5 transcription factor is a conserved evolutionary strategy to activate the genome of vertebrates. They will also help explain why Zar1 and Zar2 are so crucial for development: 1) by regulating the synthesis of Pou5 that activates transcription in embryos; 2) by regulating other mRNAs that could play other roles in embryogenesis. These studies will also build a map of genetic interactions that link the maternal and zygotic genomes.